The present invention relates to the field of agricultural biotechnology. The invention further relates to in vitro embryogenesis from somatic cells.
About one-third of the world""s sugar is refined from sugar beets. Unlike sugarcane which must be grown in tropical and subtropical regions of the world, sugar beets thrive in the temperate regions of the world. The sugar beet is the same taxonomic species (Beta vulgaris L.) as the common garden beet or red beet. Through the efforts of plant breeders, the sugar beet was bred from the garden beet by selection for increased sugar content in the root. The sugar beet breeders were able to increase the sugar content of the beet roots considerably. Compared to garden beets which have roots with a sugar content of in the range of 1-4%, the sugar content of the roots of modern sugar beet cultivars is in the range of 15-20%.
Despite the prominent position of the sugar beet in world sugar production, the rate of the development of new cultivars has been limited by a lack of efficient methods for producing transgenic sugar beets. The efforts of agricultural scientists to introduce important transgenic traits such as herbicide resistance and the resistance to diseases caused by pathogenic fungi and nematodes has been slowed by this lack of efficient methods for producing transformed sugar beet plants. While producing transformed sugar beet cells and tissues is not particularly difficult with methods such as biolistic transformation and Agrobacterium-mediated transformation, the production of transgenic sugar beet plants is limited by the lack of efficient protocols for regenerating the transformed cells and tissues into transformed sugar beet plants. Agricultural scientists need improved methods for producing transgenic sugar beet plants to provide sugar beet producers with new cultivars having the desired transgenic traits.
For some important crop species where regenerating transformed plants has proven difficult, agricultural scientists have developed methods which involve DNA delivery into isolated embryos because such embryos can be routinely regenerated into plants. Typically, either immature zygotic embryos or somatic embryos are used in such methods. A disadvantage of the use of immature zygotic embryos is that such embryos must be isolated from maternal plants at a specific developmental stage. Thus, to have a steady supply of embryos for transformation, one needs to maintain large numbers of plants of various ages. Somatic embryos can also be transformed and regenerated into transgenic plants, and are preferable over immature zygotic embryos, if relatively large numbers of somatic embryos can be produced routinely and efficiently. For important crops like soybean, repetitive embryogenic culture systems have been developed for the efficient production of somatic embryos for use in transformation (Sato et al. (1993) Plant Cell Reports 12:408-413).
Methods are provided for producing somatic embryos of Beta vulgaris. The methods find use in the agricultural biotechnology industry, particularly in methods for producing transgenic Beta vulgaris plants. The methods of the invention involve producing secondary somatic embryos comprising exposing a somatic embryo to initiation medium to induce the formation of an embryogenic tissue mass. The methods further involve isolating the embryogenic tissue mass and exposing the embryogenic tissue mass to proliferation medium to form embryogenic outgrowths. The methods further involve exposing the embryogenic outgrowths to regeneration medium to form secondary somatic embryos. The methods of the invention may additionally comprise a multiplying step to increase the number of secondary embryos produced. Such a multiplying step involves subdividing the expanded embryogenic tissue masses and exposing the subdivided tissues masses to fresh medium, particularly fresh proliferation medium. The multiplying step can be repeated one or more additional times to further increase the number of embryogenic outgrowths that can be regenerated into secondary embryos.
Methods are provided for producing Beta vulgaris plants from secondary somatic embryos. The methods involve producing secondary somatic embryos and regenerating such embryos into plants. The methods may additionally involve exposing shoots or germinated, secondary somatic embryos to rooting medium.
Methods are provided for producing transformed Beta vulgaris plants from secondary somatic embryos. The methods involve transforming a cell of a secondary somatic embryos with a gene of interest and regenerating such an embryo, or at least one transformed cell thereof, into a transformed Beta vulgaris plant.
Also provided are methods for producing callus tissues and regenerating such callus tissues into plants. The methods involve exposing seedling tissues, particularly hypocotyls and cotyledons, to a callus induction comprising at least one cytokinin, particularly thidiazuron. The methods further involve regenerating shoots that appear on callus tissues into Beta vulgaris plants.
Transformed Beta vulgaris plants, plant tissues and plant cells, and seeds thereof are also provided.